The long-term objective of this project is to overcome some of the major hurdles that prevent the clinical translation of metallic nanoparticle (NP) radiosensitization in radiation therapy (RT). Studies have shown that the passive, enhanced permeability and retention (EPR) effect itself is not sufficient to deliver the amount of intratumoral and intracellular NPs needed for in vivo radiosensitization with an affordable amount of injected NPs and the conventional NPs are cleared rapidly (~minutes) in vivo. Imaging the in vivo NP biodistribution for quantitative RT treatment planning is also an unsolved critical issue. Actively targeting and internalization into cancer cells by gadolinium (Gd) NPs conjugated to pH-Low Insertion Peptides (pHLIPs) have the potential to serve the dual purpose of enhancing uptake of NPs in tumor cells and selective, quantitative imaging by MRI. pHLIP-GdNPs can actively target solid tumors? unavoidable acidic microenvironment, which is not present in healthy tissues. Therefore, it is superior to other biomarker targeting, such as antibody targeting, which can become nonspecific and be evaded by selection of non-expressing subclones during treatment. pHLIPs can also deliver the conjugated cell-impermeable cargoes inside the cancer cells via a strong non-endocytic pathway, critical for NP-induced short-range Auger cascade and photoelectrons to reach the vital cellular targets as proved by experiments and simulations. Complementing the rapid increasing use of MRI for RT planning and on-board treatment-guidance, pHLIP-GdNPs can also solve the imageability problem for treatment plan optimization. Our preliminary MRI data shows long tumor retention of NPs (>9 hours, possibly days) post pHLIP-GdNPs injection. Specific Aims: To provide the pre-clinical foundation for more in-depth translational and clinical studies, we aim to (i) characterize pHLIP-GdNP and evaluate its RT properties in vitro; (ii) develop a mechanistic biophysical model of radiosensitization by GdNPs to elucidate relevant biolgocial mechanisms and facilitate quantitative RT treatment planning; and (iii) investigate the in vivo radiosensitization and imaging properties of pHLIP-GdNP. Research Design: (i) Characterize pHLIP-GdNP and internalization, microscopically image cellular uptake with fluorescent tags, conduct clonogenic survival experiments in cell culture with both 250 kVp and 6 MV X-rays, generate pH-dependent cell survival curves, and examine DNA damage. (ii) Use a Monte Carlo particle track structure simulation to calculate microscopic dose enhancement induced by NPs. DNA damage will be modeled to predict sensitizer enhancement ratios and compare with experimental results. (iii) Investigate the feasibility of MR imaging to determine quantitatively in vivo NP distribution and the residence-transit time in tumor and critical organs in mouse models, the enhanced radiosensitization in vivo in mice injected with pHLIP-GdNPs compared to mice injected with untargeted GdNPs using tumor growth delay assay, and the in vivo toxicity of pHLIP-GdNPs. Impact: This project can lead to a novel theranostic agent that offers improved therapeutic ratio and imageability. This new paradigm of NP delivery and imaging can a have broad impact in image-guided NP-enhanced RT.